Detection of analytes in aqueous environments

ABSTRACT

The invention relates to indicator molecules for detecting the presence or concentration of an analyte in a medium, such as a liquid, and to methods for achieving such detection. More particularly, the invention relates to copolymer macromolecules containing relatively hydrophobic indicator component monomers, and hydrophilic monomers, such that the macromolecule is capable of use in an aqueous environment.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of application Ser.No. 09/632,624, filed Aug. 4, 2000.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] This invention relates to indicator molecules for detecting thepresence or concentration of an analyte in a medium, such as a liquid,and to methods for achieving such detection. More particularly, theinvention relates to copolymer macromolecules containing relativelyhydrophobic indicator component monomers, and hydrophilic monomers, suchthat the macromolecule is capable of use in an aqueous environment.

[0005] 2. Description of the Related Art

[0006] Indicator molecules for detecting the presence or concentrationof an analyte in a medium are known. Unfortunately, many of suchindicators are insoluble or sparingly soluble in water. For example,U.S. Pat. No. 5,503,770 (James, et al.) describes a fluorescent boronicacid-containing compound that emits fluorescence of a high intensityupon binding to saccharides, including glucose. The fluorescent compoundhas a molecular structure comprising a fluorophore, at least onephenylboronic acid moiety and at least one amine-providing nitrogen atomwhere the nitrogen atom is disposed in the vicinity of the phenylboronicacid moiety so as to interact intramolecularly with the boronic acid.Such interaction thereby causes the compound to emit fluorescence uponsaccharide binding. See also T. James, et al., J. Am. Chem. Soc.117(35):8982-87 (1995). However, the compound described in example 2 ofU.S. Pat. No. 5,503,770 (having formula (6)) is substantially insolublein water, and as a practical matter requires the presence of an organicsolvent such as methanol in order to work in a liquid environment.

[0007] Lack of sufficient aqueous solubility is a severe problem whendealing with applications in an aqueous environment, for example, invivo applications. Thus, there remains a great need for adaptinginsoluble or sparingly soluble indicators for use in aqueousenvironments.

BRIEF SUMMARY OF THE INVENTION

[0008] In one aspect, the present invention is directed to an indicatormacromolecule for detecting the presence or concentration of an analytein an aqueous environment, said macromolecule comprising a copolymer of:

[0009] a) one or more indicator component monomers which individuallyare not sufficiently water soluble to permit their use in an aqueousenvironment for detecting the presence or concentration of said analyte;and

[0010] b) one or more hydrophilic monomers;

[0011] such that the macromolecule is capable of detecting the presenceor concentration of said analyte in an aqueous environment.

[0012] In another aspect, the present invention is directed to a methodfor the production of an indicator macromolecule for detecting thepresence or concentration of an analyte in an aqueous environment, saidmethod comprising copolymerizing:

[0013] a) one or more indicator component monomers which individuallyare not sufficiently water soluble to permit their use in an aqueousenvironment for detecting the presence or concentration of said analyte;and

[0014] b) one or more hydrophilic monomers;

[0015] such that the resulting macromolecule is capable of detecting thepresence or concentration of said analyte in an aqueous environment.

[0016] In another aspect, the present invention is directed to a methodfor detecting the presence or concentration of an analyte in a samplehaving an aqueous environment, said method comprising:

[0017] a) exposing the sample to an indicator macromolecule, saidmacromolecule comprising a copolymer of:

[0018] i) one or more indicator component monomers which individuallyare not sufficiently water soluble to permit their use in an aqueousenvironment for detecting the presence or concentration of said analyte;and

[0019] ii) one or more hydrophilic monomers;

[0020] such that the resulting macromolecule is capable of detecting thepresence or concentration of said analyte in an aqueous environment, andwherein the indicator macromolecule has a detectable quality thatchanges in a concentration-dependent manner when said macromolecule isexposed to said analyte; and

[0021] b) measuring any change in said detectable quality to therebydetermine the presence or concentration of said analyte in said sample.

[0022] In another aspect, the present invention provides a macromoleculewhich is capable of exhibiting an excimer effect, which comprises acopolymer of:

[0023] a) one or more excimer forming monomers, the molecularconstituents of which are capable of exhibiting an excimer effect whensuitably oriented with respect to each other; and

[0024] b) one or more other monomers;

[0025] such that the resulting macromolecule exhibits said excimereffect.

[0026] In yet another aspect, the present invention provides a methodfor producing a macromolecule which is capable of exhibiting an excimereffect, which method comprises copolymerizing:

[0027] a) one or more excimer forming monomers, the molecularconstituents of which are capable of exhibiting an excimer effect whensuitably oriented with respect to each other; and

[0028] b) one or more other monomers;

[0029] such that the resulting macromolecule exhibits said excimereffect.

[0030] In yet another aspect, the present invention provides a methodfor detecting the presence or concentration of an analyte in a sample,said method comprising:

[0031] a) exposing the sample to an indicator macromolecule, saidmacromolecule comprising a copolymer of:

[0032] i) one or more indicator component monomers, the molecules ofwhich are capable of exhibiting an excimer effect when suitably orientedwith respect to each other, and which are also capable of detecting thepresence or concentration of an analyte; and

[0033] ii) one or more other monomers;

[0034] such that the resulting macromolecule exhibits said excimereffect, and wherein the indicator macromolecule has a detectable qualitythat changes in a concentration-dependent manner when said macromoleculeis exposed to said analyte; and

[0035] b) measuring any change in said detectable quality to therebydetermine the presence or concentration of said analyte in said sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIGS. 1-2 illustrate the emission spectra of several indicatormacromolecules of the present invention as described in Example 2.

[0037]FIG. 3 depicts the synthesis of an indicator component mononerreferred to in Example 3.

[0038]FIG. 4 illustrates the normalized fluorescence emission (I/Io @427 nm) of an indicator as described in Example 5.

[0039]FIG. 5 illustrates the normalized fluorescence emission (I/Io @428 nm) of an indicator as described in Example 6.

[0040]FIG. 6 illustrates the normalized fluorescence emission (I/Io @427 nm) of an indicator as described in Example 6.

[0041]FIG. 7 illustrates the absorbance spectra of an indicator asdescribed in Example 7.

[0042] FIGS. 8-9 illustrate the ratio of the absorbance (450 nm/530 nm)of an indicator as described in Example 7.

DETAILED DESCRIPTION OF THE INVENTION

[0043] In one aspect, the present invention provides a way to utilize,in an aqueous environment, indicator components which by themselves areinsoluble or sparingly soluble in an aqueous environment. Suchindicators are, in effect, copolymerized with one or more monomers whichare sufficiently hydrophilic such that the resulting indicatormacromolecule is sufficiently hydrophilic overall so as to overcome thehydrophobic contribution of the indicator component monomers.

[0044] Suitable indicator components include indicator molecules whichare insoluble or sparingly soluble in water, and whose analyte is atleast sparingly soluble in water. Suitable analytes include glucose,fructose and other vicinal diols; α-hydroxy acids; β-keto acids; oxygen;carbon dioxide; various ions such as zinc, potassium, hydrogen (pHmeasurement), carbonate, toxins, minerals, hormones, etc. It will beappreciated that within the scope of indicator component monomer as usedherein are included mixtures of two or more individual monomers (atleast one of which is not sufficiently soluble to function adequately inan aqueous environment) which, when incorporated into the macromoleculesof the present invention, function as an indicator.

[0045] Many such indicator components are known. For example, thecompounds depicted in U.S. Pat. No. 5,503,770 are useful for detectingsaccharides such as glucose, but are sparingly soluble to insoluble inwater. Other classes of indicators include the lanthanide chelatesdisclosed in copending U.S. application Ser. No. 09/265,979 filed Mar.11, 1999 (and published as PCT International Application WO 99/46600 onSep. 16, 1999), incorporated herein by reference; polyaromatichydrocarbons and their derivatives; the indicators disclosed inco-pending application Ser. No. 09/754,217 filed Jan. 5, 2001 andapplication Ser. No. 60/269,887 filed Feb. 21, 2001, both of whichdescribe indicators having two recognition elements capable ofdiscriminating between glucose and interfering α-hydroxy acids orβ-diketones, etc.

[0046] The indicator components of the present invention will generallyhave a detectable quality that changes in a concentration-dependentmanner when the macromolecule is exposed to the analyte to be measured.Many such qualities are known and may be used in the present invention.For example, the indicator may include a luminescent (fluorescent orphosphorescent) or chemiluminescent moiety, an absorbance based moiety,etc. The indicator may include an energy donor moiety and an energyacceptor moiety, each spaced such that there is a detectable change whenthe macromolecule interacts with the analyte. The indicator may includea fluorophore and a quencher, configured such that the fluorophore isquenched by the quencher when the analyte is absent. In that situation,when the analyte is present, the indicator undergoes a configurationalchange which causes the quencher to move sufficiently distant from thefluorophore so that fluorescence is emitted. Conversely, the fluorophoreand quencher may be configured such that in the absence of analyte, theyare sufficiently separated and the fluorophore emits fluorescence; uponinteraction with the analyte, the fluorophore and quencher are moved insufficient proximity to cause quenching. The configurational changeconcept is described in more detail in co-pending application Ser. No.09/754,219, filed Jan. 5, 2001, entitled “Detection of Analytes”,incorporated herein by reference.

[0047] Other detectable moieties include those whose fluorescence isaffected by analyte interaction via photoinduced electron transfer orinductive effects. These include the lanthanide chelates disclosed incopending U.S. application Ser. No. 09/265,979 filed Mar. 11, 1999 (andpublished as PCT International Application WO 99/46600 on Sep. 16,1999), incorporated herein by reference; polyaromatic hydrocarbons andtheir derivatives; coumarins; BODIPY® (Molecular Probes, Eugene, Oreg.);dansyl; catechols; etc. Another class of moieties include those whoseabsorbance spectrum changes upon interaction of the indicator compoundwith the analyte, including Alizarin Red, etc. Another class of moietiesinclude those whose fluorescence is modulated by proximity effects,e.g., energy donor/acceptor pairs such as dansyl/dabsyl, etc.

[0048] Preferably, the detectable quality is a detectable optical orspectral change, such as changes in absorptive characteristics (e.g.,absorptivity and/or spectral shift), in fluorescent decay time(determined by time domain or frequency domain measurement), fluorescentintensity, fluorescent anisotropy or polarization; a spectral shift ofthe emission spectrum; a change in time-resolved anisotropy decay(determined by time domain or frequency domain measurement), etc.

[0049] Suitable hydrophilic monomers should be sufficiently hydrophilicso as to overcome the sum of the hydrophobic indicator componentmonomers, such that the resultant indicator macromolecule is capable offunctioning in an aqueous environment. It will be readily apparent thata wide variety of hydrophilic monomers are suitable for use in thepresent invention. For example, suitable hydrophilic monomers includemethacrylamides, methacrylates, methacrylic acid, dimethylacrylamide,TMAMA, vinyls, polysaccharides, polyamides, polyamino acids, hydrophilicsilanes or siloxanes, etc., as well as mixtures of two or more differentmonomers.

[0050] Suitable hydrophilic monomers for a given application will varyaccording to a number of factors, including intended temperature ofoperation, salinity, pH, presence and identity of other solutes, ionicstrength, etc. It would be readily apparent that the degree ofhydrophilicity of the hydrophilic monomer or the indicator macromoleculecan be increased by adding additional functional constituents such asions (e.g., sulfonate, quartenary amine, carboxyl, etc.), polar moieties(e.g., hydroxyl, sulfhydryl, amines, carbonyl, amides, etc.), halogens,etc.

[0051] It will be appreciated that the molar ratio of hydrophilicmonomer to indicator component monomer may vary widely depending on thespecific application desired. Preferred ratios of hydroplilicmonomer:indicator component monomer range from about 2:1 to about1000:1, more preferably from about 5:1 to about 50:1.

[0052] The indicator macromolecules of the present invention maygenerally be synthesized by simply copolymerizing at least one indicatorcomponent monomer with at least one hydrophilic monomer. Optimumpolymerization conditions (time, temperature, catalyst, etc.) will varyaccording to the specific reactants and the application of the finalproduct, and can easily be established by one of ordinary skill.

[0053] It will be appreciated that the indicator macromolecules of thepresent invention may have any desired extent of water solubility. Forexample, the indicator macromolecule of Examples 1 and 2 below is verysoluble, readily dissolving in aqueous solution. On the other hand,indicator macromolecules containing, for example, the hydrophilicmonomer HEMA (hydroxyethyl methacrylate) or other common hydrogelconstituents, can be non-soluble yet hydrophilic.

[0054] The soluble indicator macromolecules may be used directly insolution if so desired. On the other hand, if the desired application sorequires, the indicator macromolecule may be immobilized (such as bymechanical entrapment, covalent or ionic attachment or other means) ontoor within an insoluble surface or matrix such as glass, plastic,polymeric materials, etc. When the indicator macromolecule is entrappedwithin, for example, another polymer, the entrapping material preferablyshould be sufficiently permeable to the analyte to allow suitableinteraction between the analyte and the indicator components in themacromolecule.

[0055] Many uses exist for the indicator macromolecules of the presentinvention, including uses as indicators in the fields of energy,medicine and agriculture. For example, the indicator macromolecules canbe used as indicator molecules for detecting sub-levels or supra-levelsof glucose in blood, tissues, urine, etc., thus providing valuableinformation for diagnosing or monitoring such diseases as diabetes andadrenal insufficiency. Medical/pharmaceutical production of glucose forhuman therapeutic application requires monitoring and control.

[0056] Uses for the present invention in agriculture include detectinglevels of an analyte such as glucose in soybeans and other agriculturalproducts. Glucose must be carefully monitored in critical harvestdecisions for such high value products as wine grapes. As glucose is themost expensive carbon source and feedstock in fermentation processes,glucose monitoring for optimum reactor feed rate control is important inpower alcohol production. Reactor mixing and control of glucoseconcentration also is critical to quality control during production ofsoft drinks and fermented beverages, which consumes the largest amountsof glucose and fermentable sugars internationally.

[0057] When the indicator macromolecules incorporate fluorescentindicator substituents, various detection techniques also are known inthe art that can make use of the macromolecules of the presentinvention. For example, the macromolecules of the invention can be usedin fluorescent sensing devices (e.g., U.S. Pat. No. 5,517,313) or can bebound to polymeric materials or other substrates such as test paper forvisual inspection. This latter technique would permit, for example,glucose measurement in a manner analogous to determining pH with a stripof litmus paper. The macromolecules described herein may also beutilized as simple reagents with standard benchtop analyticalinstrumentation such as spectrofluorometers or clinical analyzers asmade by Shimadzu, Hitachi, Jasco, Beckman and others. These moleculeswould also provide analyte specific chemical/optical signal transductionfor fiber optic-based sensors and analytical fluorometers as made byOcean Optics (Dunedin, Fla.), or Oriel Optics.

[0058] U.S. Pat. No. 5,517,313, the disclosure of which is incorporatedherein by reference, describes a fluorescence sensing device in whichthe macromolecules of the present invention can be used to determine thepresence or concentration of an analyte such as glucose or other vicinaldiol compound in a liquid medium. The sensing device comprises a layeredarray of a fluorescent indicator molecule-containing matrix (hereafter“fluorescent matrix”), a high-pass filter and a photodetector. In thisdevice, a light source, preferably a light-emitting diode (“LED”), islocated at least partially within the indicator material, or in awaveguide upon which the indicator matrix is disposed, such thatincident light from the light source causes the indicator molecules tofluoresce. The high-pass filter allows emitted light to reach thephotodetector, while filtering out scattered incident light from thelight source.

[0059] The fluorescence of the indicator molecules employed in thedevice described in U.S. Pat. No. 5,517,313 is modulated, e.g.,attenuated or enhanced, by the local presence of an analyte such asglucose or other cis-diol compound.

[0060] In the sensor described in U.S. Pat. No. 5,517,313, the materialwhich contains the indicator molecule is permeable to the analyte. Thus,the analyte can diffuse into the material from the surrounding testmedium, thereby affecting the fluorescence emitted by the indicatormolecules. The light source, indicator molecule-containing material,high-pass filter and photodetector are configured such that at least aportion of the fluorescence emitted by the indicator molecules impactsthe photodetector, generating an electrical signal which is indicativeof the concentration of the analyte (e.g., glucose) in the surroundingmedium.

[0061] In accordance with other possible embodiments for using theindicator macromolecules of the present invention, sensing devices alsoare described in U.S. Pat. Nos. 5,910,661, 5,917,605 and 5,894,351, allincorporated herein by reference.

[0062] The macromolecules of the present invention can also be used inan implantable device, for example to continuously monitor an analyte invivo (such as blood or tissue glucose levels). Suitable devices aredescribed in, for example, co-pending U.S. patent application Ser. No.09/383,148 filed Aug. 26, 1999, as well as U.S. Pat. Nos. 5,833,603,6,002,954 and 6,011,984, all incorporated herein by reference.

[0063] The macromolecules of the present invention have uniqueadvantages. For example, absorbance of a sample is directly proportionalto both the concentration of the absorber and the sample path length.Thus, in an absorbance-based assay, it is apparent that for a givenlevel of absorbance, the sample path length may be greatly reduced ifthe absorber concentration is greatly increased. That desirable increasein concentration may be accomplished by decreasing the ratio of thehydrophilic monomer:indicator component monomer. In effect, the presentinvention allows the localized concentration of much more absorbercomponent into a limited space, thereby increasing the absorbance perunit thickness. Thus the present invention additionally allows use ofmuch smaller equipment when performing absorbance-based assays. It willalso be apparent that for any optically-based assay, includingfluorescence based assays, the ability to greatly increase the localconcentration of the indicator component offers several advantages. Forexample, a higher local concentration of the indicator component canpermit the utilization of thinner layers of indicator macromolecule,which in turn can greatly reduce the response time of the macromoleculeto the presence or concentration of the analyte. Further, it can resultin a higher extinction of excitation light, which can desirably reducethe incidence of autofluorescence when working in tissue systems orphysiological solutions. For example, when working with a fluorescencebased macromolecule, non-absorbed excitation light can interact with,e.g., NADH, tryptophan, tyrosine, etc. which may be present in tissue orphysiological solutions resulting in undesirable interfering fluorescentemission from those moieties. Having a high local concentration ofindicator component with high absorption can reduce that undesiredinterfering emission. Additionally, when utilizing an absorbance-basedmacromolecule in tissue or physiological solutions, it is desirable toreduce the amount of the source radiation that is reflected inpotentially varying amounts by components in surrounding tissue orfluid, such as bilirubin, e.g. Therefore, having a high localconcentration of indicator component with high absorption can reducethat undesired effect.

[0064] As a further aspect of the present invention, it has beendiscovered that certain macromolecules exhibit an excimer effect. By wayof background, when two planar molecules with aromatic structure (suchas is common with fluorophores) are concentrated to a point where theirpi electron orbital lobes may overlap, a resonance condition can thenoccur for some species where the resonance from overlap results in ahybrid (couplet) structure which is energy favorable and stable. Thesetwo planar molecules become oriented in a coplanar configuration liketwo slices of bread on a sandwich with their electron clouds overlappingbetween them. For fluorescent planar species, a characteristic downfieldemission occurs relative to the uncoupled species at wavelength ofsubstantially lower energy than the parent species. Molecules able toform such favorable resonant configurations are known as excimers. Asused herein, an excimer effect refers to the resulting characteristiclonger wavelength emission from excimers.

[0065] Some examples of typical excimer-forming polyaromatichydrocarbons include anthracene and pyrene. There are many others. Anexample is the anthracene derivative (boronate included), the indicatorcomponent used in Examples 1 and 2 of the present application. Althoughanthracene is known to form excimers in solution, one must be able toconcentrate the molecule to sufficiently high levels to observe anyexcimer character. In the case of the anthracene derivative of Examples1 and 2, the molecule is insoluble in water and insufficiently solublein a solvent such as methanol to observe excimer characteristics. In thepresent examples, the relative concentration of the anthracenederivative monomer was increased in proportion to the hydrophilicmonomer in the copolymer from 500:1, 400:1, 200:1, 100:1, 50:1, 25:1,15:1 and then 5:1. All have the characteristic blue emission at 417 nmof the anthracene derivative except at 5:1 ratio, a green emissionsuddenly appears. This green emission is that of an excimer hybrid andthe emission has been shifted downfield by approximately 100+ nanometers(˜515-570 nm, green). The concentration of the overall solution does notneed to be high since the distance between planar species is beingcontrolled by placement along the polymer backbone rather than solubleconcentration in 3-D space.

[0066] Surprisingly, it has been found that the excimer emission regionis not responsive to changes in analyte concentration, but is responsiveto all other aspects of the system analyzed, such as excitationintensity, temperature, and pH. As a result, the present indicatormacromolecules may serve as both an indicator and an internal reference.For example, an ideal referencing scheme is one where the emissionintensity at an indicator wavelength (i.e., the wavelength influenced bythe analyte) is divided optically using select bandpass filters, by theemission intensity at the excimer wavelength. The resultant valuecorrects for interfering factors which affect fluorescent emissionproperties, such as fluorescent quenching by, e.g., oxygen, drift anderror in pH, power factors and drift affecting LED intensity, ambienttemperature excursions, etc.

[0067] It will be readily appreciated that the macromolecules of thepresent invention which exhibit an excimer effect will be useful in bothaqueous and non-aqueous environments. Consequently, thosemacromolecules, as well as the component monomers (excimer-forming andother monomer), may range from hydrophilic to hydrophobic, dependingupon the desired application.

[0068] Also, when the excimer macromolecules of the present inventionare used to detect the presence or concentration of an analyte, themacromolecule may be used directly in solution, or may be immobilized asdescribed above.

[0069] The macromolecules of the present invention can be prepared bypersons skilled in the art without an undue amount of experimentationusing readily known reaction mechanisms and reagents, including reactionmechanisms which are consistent with the general procedures describedbelow.

EXAMPLE 1

[0070] a) Preparation of 9-[(methacryloylaminopropylamino)methyl]anthracene

[0071] (A) One-Phase

[0072] To a suspension of N-(3-aminopropyl) methacrylamide hydrochloride(Polysciences, #21200) (11.82 g, 0.066 mole, 3.0 eq) and a trace ofinhibitor DBMP (2,6-di-t-butyl-4-methylphenol) (10 mg) in chloroform(250 mL) stirring in an ice-water bath, diisopropylethylamine (25 mL,18.55 g. 0.144 mole, 6.5 eq) was added dropwise in 20 minutes. Themixture was allowed to warm up to room temperature and cooled again inice-water bath. A clear solution of 9-chloromethylanthracene (5.0 g,0.022 mole) in chloroform (100 mL) was added dropwise over 1 hour. Itwas run at 25° C. for 1 hour, 50° C. for 12 hours and then 70° C. for 2hours.

[0073] The mixture was washed with water (60 mL×4), and the aqueouslayer was extracted with methylene chloride. The organic layers werecombined, dried over Na₂SO₄, separated, and the solvent was removedunder reduced pressure at 40° C. The crude material was thenchromatographed on silica gel with 2-5% methanol in methylene chlorideto give 2.44 g (33.4%) of product as a solid. TLC (silica gel): R_(f)0.39 (MeOH/CH₂Cl₂=1/9), a single spot.

[0074] (B) Two-Phase

[0075] To a clear solution of N-(3-aminopropyl) methacrylamidehydrochloride (788 mg, 4.41 mmole, 10 eq) and a trace of inhibitor MEHQ(methylether hydroquinone) (2 mg) in a mixture of water (30 mL) andtetrahydrofuran (30 mL) stirring in an ice-water bath. A Na₂CO₃/NaHCO₃buffer (66 mL, 0.2 M, pH 10) was added in 1 hour and a solution of9-chloromethylanthracene (100 mg, 0.441 mmole) in chloroform (100 mL)was added in 3 hours. It was run at 25° C. for 7 hours and then 55° C.for 12 hours.

[0076] The organic layer was separated, washed with water (50 mL×4), andthe aqueous layers were extracted with methylene chloride. The organiclayers were combined, dried over Na₂SO₄, separated, and the solvent wasremoved with reduced pressure at 45° C. The crude material (270 mg) wasthen chromatographed on silica gel with 10-20% methanol in methylenechloride to give 28.7 mg (19.6% of product as a solid TLC (silica gel):R_(f) 0.77 (MeOH/CH₂Cl₂=3/7), a single spot.

[0077] b) Preparation of9-[[N-methacryloylaminopropyl-N-(o-boronobenzyl)amino]methyl]anthracene

[0078] To a solution of the product obtained in step a) above (2.440 g,0.00734 mole) and a trace of inhibitor DBMP (10 mg) in chloroform (200mL) stirring in an ice-water bath, DIEA (diisopropylethylamine) (2.846g, 3.84 mL, 0.022 mole, 3.0 eq) was added by portions in 10 minutes, andthen a solution of2,2-dimethylpropane-1,3-diyl[o-(bromomethyl)phenyl]boronate (2.492 g,0.00881 mole, 1.2 eq) in chloroform (15 mL) was added in 30 minutes. Thereaction was run at room temperature for 20 hours.

[0079] The mixture was washed with water, separated and the aqueouslayers were extracted with methylene chloride. The organic layers werecombined, dried over Na₂SO₄, separated and the solvent was removed withreduced pressure at 25° C. The semi-solid (4.75 g) was thenchromatographed on silica gel with 2-5% methanol in methylene chlorideto give 2.50 g (76.3%) of product as a lightly yellow crystalline solid,mp 72-73° C., TLC (silica gel): R_(f) 0.36 (MeOH/CH₂Cl₂=1/9). It issoluble in CH₂Cl₂, CHCl₃, THF, CH₃OH, and C₂H₅OH. Limited solubility inH₂O and ether.

[0080] c) Preparation of water-soluble-copolymeric solutions of MAPTACand9-[[N-methacryloylaminopropyl-N-(o-boronobenzyl)amino]methyl]anthracene

[0081] (50:1) solution: To a solution of the monomer (42.3 mg, 0.0908mmole) in ethanol (100%, 1.5 mL), MAPTAC[3-(methacryloylamino)propyl]trimethylammonium chloride (2.0 mL, 1.0 g,4.54 mmole, 50 eq) and an AIBN (azobisisobutyl nitrile) ethanolicsolution (0.183 M, 0.2 mL) as radical initiator were added, a clearsolution was obtained. It was saturated with nitrogen and then heated to70° C. in 1 hour, and kept at 70° C. for 80 minutes, and a viscousliquid was obtained.

[0082] The liquid obtained was treated with water (26 mL) and filteredthrough a microfilter (0.45 um) to give a clear solution. After dialysisthrough a cellulose acetate membrane (MWCO 3500) with water 5 L×4), itwas concentrated with polyethylene glycol (MW 20 K) to a clear solution(34.54 g). Concentration: 24.0 mg solid in 1.0 g solution, total solid829 mg, yield 79.5%.

[0083] Similar procedures were applied to prepare copolymeric solutionsof 500:1, 400:1, 200:1, 100:1, 50:1, 25:1, 15:1, and 5:1 molar ratios ofhydrophilic monomer:indicator.

[0084] Glucose Modulation of 50:1 and 25:1 Co-Polymers

[0085] The modulation of the fluorescence of the 50:1 and 25:1 indicatormacromolecules by glucose solutions having various concentrations isshown below in Tables 1 and 2. Table 1 shows the results using twodifferent concentrations (15 mg/ml and 25 mg/ml) of the 25:1 indicatormacromolecule of this example with four different glucoseconcentrations. Table 2 shows the results using two differentconcentrations (10 mg/ml and 20 mg/ml) of the 50:1 indicatormacromolecule of this example with four different glucoseconcentrations. In both Tables, I/Io is the ratio of the emittedintensities at 420 nm after and before exposure to glucose (365 nmexcitation). TABLE 1 I/Io for 15 mg/ml I/Io for 25 mg/ml Glucoseindicator indicator concentration macromolecule macromolecule (mM)(25:1) (25:1) 0 1.00 1.00 50 1.44 1.50 100 1.75 1.90 200 2.13 2.33

[0086] TABLE 2 I/Io for 10 mg/ml I/Io for 20 mg/ml Glucose indicatorindicator concentration macromolecule macromolecule (mM) (50:1) (50:1) 01.00 1.00 50 1.40 1.48 100 1.70 1.79 200 2.04 2.22

EXAMPLE 2

[0087] This example demonstrates a surprising and useful excimer effectpresent in connection with the 5:1 indicator macromolecule prepared inExample 1.

[0088]FIG. 1 depicts the emission spectra of the 5:1 indicatormacromolecule when exposed to three concentrations of glucose (0 mM, 30mM and 60 mM) after excitement by light at 365 nm. Also shown in theshaded region of FIG. 1 is the emission of the non-excimer 25:1indicator macromolecule from Example 1. The excimer emission regionshows an “isosbestic region” rather than an isosbestic point. It can beseen from FIG. 1 that the excimer emission region (the region where the0 mM, 30 mM and 60 mM glucose lines overlap) is not responsive tochanges in glucose concentration (just like an isosbestic point). Theexcimer emission region begins approximately 100 nm downfield from thepeak responsive wavelength of the anthracene derivative modulation.Except for glucose, the excimer is responsive to all other aspects ofthe system, such as excitation intensity, temperature, and pH.Therefore, an ideal referencing scheme is one where the amplitude orsignal value at 415 nm is divided electronically by the amplitude orsignal value at 515 nm or another wavelength or range of wavelengthswithin the excimer emission region, and the resultant value will becorrected for drift and error in pH, power factors and drift affectingLED intensity, ambient temperature excursions, etc. That is demonstratedbelow.

[0089] Demonstration of Excitation Intensity, Temperature and pHCorrection

[0090] The glucose modulation of the 5:1 indicator macromolecule wasmeasured with three different glucose solutions (0 mM, 100 mM and 200mM). The emission spectra were determined for each of the glucosesolutions at three different spectrophotometer slit configurations forsource and emitted light (1.5 being narrower and 3 being wider). Thedata are shown in Table 3. In the Table, the ratio of the emissionintensity at 420 nm to the emission intensity at 550 nm is relativelyindependent of slit configuration. TABLE 3 I420/I550 I420/I550 SlitI420/I550 100 mN 200 mM Configuration 0 mN glucose glucose glucose1.5/1.5 3.92 6.18 7.36 1.5/3   3.93 6.12 7.25 3/3 4.00 6.27 7.28

[0091] The temperature stability of the 5:1 excimer indicatormacromolecule was determined. The ratio of the emissions at 420 nm and550 nm for a 1 mg/ml solution of the 5:1 excimer indicator macromoleculeexposed to 200 mM glucose (pH 7.5) was 7.57 at room temperature and 7.53at approximately 60° C.

[0092] The pH stability of the 5:1 excimer indicator macromolecule wasalso determined. The ratio of the emissions at 420 nm and 550 nm for a 1mg/ml solution of the 5:1 excimer indicator macromolecule at threedifferent pH levels (6.5, 7.0 and 7.5) were determined (excitation lightat 370 nm, slits 1.5,3), and are shown in Table 4. The full emissionspectra are shown in FIG. 2. The variation over the range tested wasstatistically insignificant. TABLE 4 I420/I550 pH 6.5 I420/I550 pH 7.0I420/I550 pH 7.5 4.28 ± 0.18 4.60 ± 0.37 4.29 ± 0.19

[0093] It is believed that the stability of the excimer complex(presumably through the pi cloud) exceeds that of the non-excimeranthracene derivative, and, that the boronate recognition feature, whichis able to perturb the properties of the non-excimer, and thus make agood indicator, is not able to perturb the more stable excimer complexand thus the excimer makes a very good reference indicator. Thereference molecule is structurally unaltered from the read channelindicator. The polymer matrix may be the same, and in this example is infact the same macromolecule. The recognition element is open and intact,but the inductive energy influence between recognition element andfluorophore center has been muted.

[0094] The foregoing is quite significant, because it can eliminate theneed for separate physical and/or chemical environments betweenindicator and reference molecules.

EXAMPLE 3

[0095] The synthesis of a suitable lanthanide chelate indicatorcomponent monomer is depicted in FIG. 3. Compounds (1) and (2) arecommercially available from Macrocyclics, Richardson, Tex. (compound (2)is known as p-NH₂-Bz-DOTA). The end product (9) may be co-polymerizedwith one or more other monomers to form an indicator macromolecule.

EXAMPLE 4

[0096] Single-Methacrylamide Monomer of Bis-Boronate-Anthracene

[0097] A.9-chloromethyl-10-[[2-(2-hydroxyethoxy)ethylamino]-methyl]anthracenehydrochloride salt.

[0098] To a suspension of 9,10-bis(chloromethyl)anthracene (5.18 g, 18.8mmole, 3.99 equiv.) in 200 mL of NMP was added 2-(2-aminoethoxy)ethanol(0.495 g, 0.475 mL, 4.71 mmole). The mixture was stirred in the dark for17 hours. At this time, the reaction mixture was concentrated to ˜50 mLunder vacuum at 50° C. The residue was purified by silica gel chromatography (150 g gravity grade silica gel, 0-10% CH₃OH/CH₂Cl₂) to yield0.425 g (24%) of a yellow/orange solid.

[0099] TLC: Merck silica gel 60 plates, R_(f) 0.72 with 70/30CH₂Cl₂/CH₃OH, see with UV (254/366), ninhydrin stain.

[0100] HPLC: HP 1100 HPLC chromatograph, Vydac 201TP 10×250 mm column,0.100 mL injection, 2 mL/min, 370 nm detection, A=water (0.1% HFBA) andB=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18 min, 80-100%B over 2 min, 100% B 2 min, retention time 16.1 min.

[0101] B.9-[[2-(2-hydroxyethoxy)ethylamino]methyl]-10-[[(3-methacrylamido)propylamino]methyl]anthracene.

[0102] To a suspension of N-(3-aminopropyl)methacrylamide hydrochloridesalt (3.08 g, 17.2 mmole, 4.2 equiv.), DIEA (5.19 g, 7.00 mL, 40.1mmole, 9.8 equiv.) and ˜3 mg of BHT in 125 mL CHCl₃ at 23° C. was addeddropwise a solution of9-chloromethyl-10-[[2-(2-hydroxyethoxy)ethylamino]-methyl]anthracenehydrochloride salt (1.56 g, 4.10 mmole) in 25 mL of CHCl₃. The mixturewas subsequently stirred in the dark for 92 hours. At this time, thereaction mixture was filtered and washed with 2×40 mL of NaHCO₃(saturated aqueous solution). The organic extract was dried overanhydrous Na₂SO₄, filtered and concentrated to yield a sticky orangesolid which was purified by alumina chromatography (50 g activatedneutral alumina, 0-5% CH₃OH/CH₂Cl₂) to yield 0.364 g (20%) of an orangesolid.

[0103] TLC: Merck silica gel 60 plates, Rf 0.16 with 70/30 CH₂Cl₂/CH₃OH,see with UV (254/366), ninhydrin stain.

[0104] HPLC: HP 1100 HPLC chromatograph, Vydac 201TP 10×250 mm column,0.100 mL injection, 2 mL/min, 370 nm detection, A=water (0.1% HFBA) andB=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18 min, 80-100%B over 2 min, 100% B 2 min, retention time 16.85 min.

[0105] C.9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)-ethylamino]methyl]anthracene.(Single-methacrylamide monomer).

[0106] A solution of9-[[2-(2-hydroxyethoxy)ethylamino]-methyl]-10-[[(3-methacrylamido)propylamino]methyl]-anthracene(0.343 g, 0.763 mmole), DIEA (0.965 g, 1.30 mL, 9.8 equiv.) and(2-bromomethylphenyl)boronic acid neopentyl ester (1.09 g, 3.85 mmole,5.0 equiv.) in 20 mL CHCl₃ at 23° C. was stirred in the dark for 25hours. At this time, the reaction mixture was concentrated initially byrotary evaporation, then using a vacuum pump to remove DIEA. The residuewas purified by alumina column chromatography (40 g activated neutralalumina, 0-10% CH₃OH/CH₂Cl₂) to yield 0.299 g (46%) of a yellow orangesolid. This product may be copolymerized with one or more other monomersto form an indicator macromolecule. The boronate groups should bedeprotected prior to use.

[0107] FAB MS: Calc'd for C₅₁H₆₅B₂N₃O₇ [M]⁺ 854; Found [M+1]⁺ 855.

[0108] TLC: Merck basic alumina plates, Rf 0.35 with 95/5 CH₂Cl₂/CH₃OH,see with UV (254/366).

[0109] HPLC: HP 1100 HPLC chromatograph,Vydac 201TP 10×250 mm column,0.100 mL injection, 2 mL/min, 370 nm detection, A=water (0.1% HFBA) andB=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18 min, 80-100%B over 2 min, 100% B 2 min, retention time 19.7 min.

EXAMPLE 5

[0110] Dual-Methacrylamide Monomer of Bis-Boronate-Anthracene

[0111] A. 9,10-bis[3-(methacrylamido)propylamino]methylanthracene.

[0112] A suspension of 9,10-bis(chloromethyl)anthracene (1.5 g, 5.45mmole), DIEA (28.17 g, 38.00 mL, 218 mmole, 40 equiv.),N-(3-aminopropyl)methacrylamide hydrochloride salt (9.76 g, 54.5 mmole,10.0 equiv.), and ˜5 mg of BHT in 200 mL CHCl₃ at 23° C. was stirred inthe dark for 4 days at 40° C. At this time, the temperature wasincreased to 45° C. and the mixture was stirred for 3 days longer. Atthis time, a precipitate had formed. The mixture was filtered, and thesolid product dissolved in the minimum amount of CH₂Cl₂. A yellowcrystalline solid, the bis hydrochloride salt of the desired product,formed overnight (3.15 g, quantitative).

[0113] TLC: Merck basic alumina plates, Rf 0.31 with 90/10 CH₂Cl₂/CH₃OH,see with UV (254/366).

[0114] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.100 mL injection, 0.75 mL/min, 360 nm detection, A=water (0.1%HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18min, 80-100% B over 2 min, 100% B 2 min, retention time 15.0 min.

[0115] B.9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methylanthracene.

[0116] A solution of9,10-bis[3-(methacrylamido)-propylamino]methylanthracene (0.650 g, 1.34mmole of the free amine), DIEA (0.612 g, 0.825 mL, 4.74 mmole, 3.55equiv.), (2-bromomethylphenyl)boronic acid neopentyl ester (1.34 g, 4.74mmole, 3.55 equiv.) and BHT (5 mg as inhibitor) in 20 mL CHCl₃ at 23° C.was stirred in the dark for 5 days. At this time, the reaction mixturewas concentrated in vacuo and the residue was purified by aluminachromatography (200 g activated neutral alumina, 0-2% CH₃OH/CH₂Cl₂) toyield 0.465 g (39%) of a very viscous yellow oil.

[0117] TLC: Merck basic alumina plates, Rf 0.59 with 90/10 CH₂Cl₂/CH₃OH,see with UV (254/366).

[0118] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.050 mL injection, 0.75 mL/min, 360 nm detection, A=water (0.1%HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18min, 80-100% B over 2 min, 100% B 2 min, retention time 16.9 min.

[0119] C. Preparation of N,N-Dimethylacrylamide Hydrogel with GlucoseIndicator:

[0120] A solution of N,N-dimethylacrylamide (40% wt.) andN,N′-methylenebisacrylamide (0.8% wt.) in ethylene glycol was prepared.9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)-benzyl]-N-[3-(methacrylamido)propylamino]methylanthracene(17.8 mg, 2×10⁻⁵ mole) and 40 μL of aqueous ammonium persulfate (5% wt)were combined with 1 mL of ethylene glycol monomer solution. Theresulting solution was placed in a glove box purged with nitrogen. Anaqueous solution of N,N,N′,N′-tetramethylethylenediamine (80 μL, 5% wt.)was added to the monomer formulation to accelerate polymerization. Theresulting formulation was poured in a mold constructed from microscopeslides and 100 micron stainless steel spacer. After being kept for 8hours in nitrogen atmosphere the mold was placed in phosphate bufferedsaline (PBS) (10 mM PBS, pH=7.4), the microscope slides were separated,and the hydrogel was removed. The hydrogel was washed with 100 mL of PBScontaining 1 mM lauryl sulfate sodium salt and 1 mM EDTA sodium salt for3 days, the solution being changed every day, followed by washing withDMF/PBS (10/90 by vol., 3×100 mL), and finally with PBS (pH=7.4, 3×100mL). The resulting hydrogel polymer was stored in PBS (10 mM PBS,pH=7.4) containing 0.2% wt. sodium azide and 1 mM EDTA sodium salt.

[0121] D. Modulation of Fluorescence With Glucose, Lactate andAcetoacetate

[0122] The modulation of the fluorescence of the indicator macromolecule(which contains two recognition elements) prepared in this example byglucose, lactate and acetoacetate was determined. FIG. 4 shows thenormalized fluorescence emission (I/Io @ 427 nm) of the hydrogel of thisexample in 10 mM PBS, pH 7.4 containing 0.2% NaN₃ and 1 mM EDTAcontaining various amounts of sodium-L-lactate, lithium acetoacetate orα-D-glucose. Data were recorded using a Shimadzu RF-5301spectrofluorometer with excitation @365 nm (slit=3 nm) and emission at427 nm (slit=3 nm) at low sensitivity at 37° C. using a temperaturecontrolled sample holder. The cuvettes containing 3 mL of the desiredsolution were equilibrated at 37° C. for 15 minutes before measurement.Each hydrogel sample was measured in four independent samples. Errorbars are standard deviation with quadruplicate values for each datapoint. The hydrogels containing a glucose recognition molecule wereprepared as previously described. The hydrogels were mounted on glassslides and covered with polyester mesh in PMMA cuvettes at 45° to theincident light. Solutions of 1, 5, 10 and 20 mM sodium L-lactate[Aldrich], 5, 10 and 20 mM lithium acetoacetate [Aldrich], and 1, 2, 4,5, 10, and 20 mM α-D-glucose were prepared in 10 mM PBS, pH 7.4containing 0.2% NaN₃ and 1 mM EDTA. The fluorescence of the copolymerwas affected by the presence of glucose, but not by the presence oflactate or acetoacetate.

EXAMPLE 6

[0123] Single- and Dual-Methacrylate Monomers of Bis-Boronate-Anthracene

[0124] A. 9,10-bis[[2-(2-hydroxyethoxy)ethylamino]methyl]-anthracene.

[0125] To a solution of 2-(2-aminoethoxy)ethanol (31.4 g, 30.0 mL, 299mmole, 20.9 equiv.) in 40 mL CHCl₃ at 23° C. was added9,10-bis(chloromethyl)anthracene (3.94 g, 14.3 mmole). The solution wasstirred in the dark for 67 hours. At this time, 100 mL CH₂Cl₂ were addedand the solution was washed with 1×50 mL and 2×100 mL portions of NaHCO₃(saturated aqueous solution). The organic extract was dried overanhydrous Na₂SO₄, filtered and concentrated to yield 4.67 g (79%) of ayellow powder. Product (˜85 % pure by RP-HPLC) was carried on as is.

[0126] HPLC conditions: HP 1100 HPLC chromatograph, Vydac 201TP 10×250mm column, 0.100 mL injection, 2 mL/min, 370 nm detection, A=water (0.1%HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18min, 80-100% B over 2 min, 100% B 2 min, retention time 15.6 min.

[0127] B.9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene.

[0128] A solution of9,10-bis[[2-(2-hydroxyethoxy)ethylamino]-methyl]anthracene (4.02 g, 9.75mmole), DIEA (12.6 g, 17.0 mL, 97.5 mmole, 10.0 equiv.) and(2-bromomethylphenyl)boronic acid neopentyl ester (13.7 g, 48 mmole, 4.9equiv.) in 125 mL CHCl₃ at 23° C. was stirred in the dark for 46 hours.At this time, the reaction mixture was concentrated initially by rotaryevaporation, then using a vacuum pump to remove the DIEA. The residuewas purified by alumina column chromatography (150 g activated neutralalumina, 0-3% CH₃OH/CH₂Cl₂) to yield 5.67 g (70%) of a viscous oil whichsolidified upon standing. Product (˜85% pure by RP-HPLC) was carried onas is.

[0129] TLC: Merck basic alumina plates, Rf 0.33 with 95/5 CH₂Cl₂/CH₃OH,see with UV (254/366).

[0130] HPLC conditions: HP 1100 HPLC chromatograph, Vydac 201TP 10×250mm column, 0.100 mL injection, 2 mL/min, 370 nm detection, A=water (0.1%HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18min, 80-100% B over 2 min, 100% B 2 min, retention time 18.8 min.

[0131] C.9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)-ethylamino]methyl]anthracene.(Single-methacrylate monomer).

[0132] A solution of9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]-anthracene(0.298 g, 0.359 mmole), methacrylic acid (0.304 g, 0.300 mL, 3.53 mmole,9.84 equiv.), DCC (0.965 g, 4.68 mmole, 13.0 equiv.) andN,N-dimethylamino-pyridine (0.020 g, 0.16 mmole, 0.46 equiv.) in 15 mLCH₂Cl₂ at 23° C. was stirred in the dark for 4 hours. At this time, thereaction mixture was filtered and concentrated by rotary evaporation.The residue was purified by alumina column chromatography (50 gactivated neutral alumina, 0-4% CH₃OH/CH₂Cl₂) to yield 0.150 g (47%) ofa yellow solid.

[0133] FAB MS: Calc'd for C₅₂H₆₆B₂N₂O₉ [M]⁺ 885; Found [M+1]⁺ 886.

[0134] TLC: Merck basic alumina plates, Rf 0.45 with 95/5 CH₂Cl₂/CH₃OH,see with UV (254/366).

[0135] HPLC: HP 1100 HPLC chromatograph, Vydac 201TP 10×250 mm column,0.100 mL injection, 2 mL/min, 370 nm detection, A=water (0.1% HFBA) andB=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18 min, 80-100%B over 2 min, 100% B 2 min, retention time 21 min.

[0136] D. Water Soluble Copolymer of9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]-anthraceneand TMAMA (1:50 molar ratio).

[0137] To a solution of [2-(methacryloxy)ethyl]trimethyl-ammoniumchloride (TMAMA, 70 wt % aqueous solution, 0.344 g monomer, 1.66 mmole,50 equiv.) in 0.600 mL water was added a solution of9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]-methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene(0.0024 g, 0.0033 mmole) in 3.00 mL MeOH. To this mixture was added4,4′-azobis(4-cyanovaleric acid) (0.0075 g, 0.027 mmole, 1.6 mole % oftotal monomer). The solution was filtered through a 0.45 μ membranefilter, was purged with nitrogen gas and then heated in the dark at 55°C. for 16 hours. At this time, the viscous solution was cooled to 25° C.and concentrated in vacuo. The residue was diluted with 20 mL water andfiltered through a 0.2 μ membrane filter. The polymer solution wasdialyzed through a cellulose acetate membrane (MWCO 3500) against 2×4 Lof water. From the dialysis was obtained 38.5 mL of polymer solution.Concentration of a portion of this solution to dryness indicated 0.0075g polymer per 1.0 mL solution. Overall 0.289 g (77%) yield of polymer.

[0138] E. Modulation of Fluorescence With Glucose, Lactate andAcetoacetate

[0139] The modulation of the fluorescence of the copolymer (whichcontains two recognition elements) prepared in step D of this example byglucose, lactate and acetoacetate was determined. FIG. 5 shows thenormalized fluorescence emission (I/Io @ 428 nm) of a 1.5 mg/mL solutionof anthracene bis boronate-TMAMA (1:50 mole ratio) copolymer in PBScontaining a) 0-20 mM glucose; b) 0-20 mM lactate; c) 0-20 mM lithiumacetoacetate. Spectra were recorded using a Shimadzu RF-5301spectrafluorometer with excitation @365 nm; excitation slits at 1.5 nm;emission slits at 1.5 nm; ambient temperature. The fluorescence of thecopolymer was affected by the presence of glucose, but not by thepresence of lactate or acetoacetate.

[0140] F.9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]anthracene.(Dual-methacrylate monomer).

[0141] A solution of9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene(0.100 g, 0.120 mmole), methacrylic acid (0.112 g, 0.110 mL, 1.30 mmole,10.8 equiv.), DCC (0.316 g, 1.53 mmole, 12.8 equiv.) andN,N-dimethylamino-pyridine (0.014 g, 0.11 mmole, 0.92 equiv.) in 5 mLCH₂Cl₂ was stirred at 0° C. for 1 hour, then 23° C. for 22 hours. Atthis time, the reaction mixture was filtered and concentrated by rotaryevaporation. The residue was purified by alumina column chromatography(30 g activated neutral alumina, 0-2% CH₃OH/CH₂Cl₂) to yield 0.030 g(26%) of a yellow solid. This product may be copolymerized with one ormore other monomers to form an indicator macromolecule. The boronategroups should be deprotected prior to use.

[0142] FAB MS: Calc'd for C₅₆H₇₀B₂N₂O₁₀ [M]⁺ 953; Found [M]⁺ 951 (weakmolecular ion peak).

[0143] TLC: Merck basic alumina plates, Rf 0.67 with 95/5 CH₂Cl₂/CH₃OH,see with UV (254/366).

[0144] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.100 mL injection, 0.75 mL/min, 2 mL injection loop, 370 nmdetection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min, retentiontime 19.6 min.

[0145] G. Preparation of HEMA/SPE/MAA Hydrogel with Glucose Indicator:

[0146] A solution of hydroxyethyl methacrylate (HEMA, 0.078 mL, 0.084 g,0.64 mmol), methacrylic acid (MAA, 0.030 mL, 0.030 g, 0.35 mmol),polyethyleneglycol dimethacrylate 1000 (PEGDMA, 0.5 mg/mL aqueoussolution, 0.048 mL), andN,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)-ammonium-batain (SPE,0.462 g, 1.65 mmol) in 0.900 mL of ethylene glycol was prepared.9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]anthracene(0.0096 g, 0.010 mmol) and 0.020 mL of 5% wt. aqueous solution ofammonium persulfate were combined with 0.500 mL of the ethylene glycolmonomer solution. This solution was placed in glove box purged withnitrogen. An aqueous solution of N,N,N′,N′-tetramethylethylenediamine(0.040 mL, 5% wt.) was added to the monomer formulation to acceleratepolymerization. The resulting formulation was poured in a moldconstructed from microscope slides and a 100 micron stainless steelspacer. After being kept for 8 hours in nitrogen atmosphere the mold wasplaced in phosphate buffered saline (PBS, pH=7.4), the microscope slideswere separated, and the hydrogel was removed. The hydrogel was washedwith 100 mL of PBS containing 1 mM lauryl sulfate sodium salt for 3days, the solution being changed every day, followed by washing withMeOH/PBS (20/80 by vol. 3×100 mL), and finally with PBS (3×100 ML). Theresulting hydrogel polymer was stored in PBS (pH=7.4) containing 0.2%wt. sodium azide and 1 mM EDTA sodium salt.

[0147] H. Modulation of Fluorescence with Glucose:

[0148] The modulation of the fluorescence of the dual methacrylateindicator compound prepared in this example by glucose was determined.FIG. 6 shows the relative fluorescence emission (I@427 nm) of a HEMA/SPEhydrogel (100 micron thickness, prepared as previously described)containing the dual methacrylate glucose recognition molecule of thisexample in PBS (pH 7.4 containing 0.2% NaN₃ and I mM EDTA) containing 0to 20 mM α-D-glucose. The hydrogels were mounted on glass slides andcovered with black polyester mesh (so far America, Depew, N.Y.) in PMMAcuvettes at 45° to the incident light. All measurements were made at 37°C. in a Shimadzu RF-5301 spectrofluorometer with excitation at 365 nm(slit=1.5 nm) and emission at 427 nm (slit=1.5 nm) at high sensitivityusing a temperature controlled sample holder. The cuvettes containing 3mL of the desired glucose solution (0, 1, 2, 4, 5, 10, 20 mM glucose)were equilibrated at 37 C. for 30 minutes before measurement. A singleexponential function was used to fit the raw fluorescence data.

EXAMPLE 7

[0149] Effect of glucose or lactate on acrylamide gel containingN-[3-(methacrylamido)propyl]-3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonamide(Alizarin Red S monomer) andα,α′-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-1,4-xylene(bis boronic acid monomer):

[0150] A. 3,4-Dihydroxy-9,10-dioxo-2-anthracenesulfonyl chloride:

[0151] 3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonic acid sodium salt(1.4 g, 3.9 mmoles) was combined with 30 mL of chlorosulfonic acid andheated to 90° C. for 5 hours, after which the solution was cooled to 0°C. and poured into 100 g of ice. After the ice melted the solution wasextracted with CH₂Cl₂ (3×100 mL), methylene chloride extracts werecombined, dried with Na₂SO₄ and evaporated to produce 0.87 g of solid(Yield 66%).

[0152] B.N-[3-(methacrylamido)propyl]-3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonamide:

[0153] 3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonyl chloride (96 mg,0.28 mmoles) and N-(3-aminopropyl) methacrylamide hydrochloride (108 mg,0.6 mmoles) were combined with 20 mL of CH₂Cl₂. To this suspension Et₃N(303 mg, 3 mmoles) was added. The mixture was stirred at roomtemperature for 24 hours, filtered, and solvent was evaporated. Theresulting solid was subjected to column chomatography on SiO₂ (10 g)with CH₂Cl₂/MeOH (90/10) as an eluent. The product was obtained as a redsolid (80 mg, 64% yield).

[0154] FAB MS: Calculated for C₂₁H₂₀N₂O₇S M⁺ 445; Found M⁺ 445.

[0155] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.100 mL injection, 0.75 mL/min, 2 mL injection loop, 370 nmdetection, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2min, 10-80% B over 18 min, 80-100% B over 2 min, 100% B 2 min, retentiontime 17.67 min.

[0156] C. α,α′-bis[3-(methacrylamido)propylamino]-1,4-xylene.

[0157] A solution of N-(3-aminopropyl)methacrylamide hydrochloride salt(3.00 g, 16.8 mmole, 2.21 equiv.), DIEA (6.5 g, 8.8 mL, 50 mmole, 6.6equiv.), terephthaldicarboxaldehyde (1.02 g, 7.60 mmole) and Na₂SO₄(10.7 g, 75.3 mmole, 9.91 equiv.) in 75 mL anhydrous MeOH was stirred inthe dark at 25° C. for 18 hours. At this time, more Na₂SO₄ (10.7 g, 75.3mmole, 9.91 equiv.) was added and stirring continued for 6 hours longer.At this time, the solution was filtered and NaBH₄ (1.73 g, 45.7 mmole,6.01 equiv.) was added to the filtrate in portions and subsequentlystirred at 25° C. for 21 hours. The suspension was filtered throughCelite and the filtrate was concentrated. The residue was dissolved in100 mL CH₂Cl₂ and washed 1×25 mL saturated aqueous NaHCO₃. The organicextract was dried over anhydrous Na₂SO₄, filtered and concentrated toyield a viscous oil. The product was carried on as is.

[0158] HPLC: HP 1100 HPLC chromatograph, Vydac 201TP 10×250 mm column,0.100 mL injection, 2.00 mL/min, 260 nm detection, A=water (0.1% HFBA)and B=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18 min,80-100% B over 2 min, 100% B 2 min, retention time 15.8 min.

[0159] D.α,α′-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-1,4-xylene.

[0160] A solution of α,α′-bis[3-(methacrylamido)-propylamino]-1,4-xylene(2.94 g, 7.61 mmole), DIEA (2.97 g, 4.00 mL, 23.0 mmoles, 3.02 equiv.),(2-bromomethyl-phenyl)boronic acid neopentyl ester (6.50 g, 23.0 mmole,3.02 equiv.) and BHT (5 mg as inhibitor) in 75 mL CH₂Cl₂ at 25° C. wasstirred in the dark for 28 hours. At this time, the mixture was washed1×25 mL saturated aqueous NaHCO₃. The organic extract was dried overanhydrous Na₂SO₄, filtered and concentrated. To the residue was added200 mL ether and the suspension was stirred for 18 hours. The suspensionwas filtered and the residue dissolved in CH₂Cl₂, filtered and thefiltrate concentrated. To the solid residue was added 150 mL ether andthe suspension was stirred for 18 hours. At this time, the suspensionwas filtered yielding 1.98 g (33%) of a fluffy pink powder, which had amaximum solubility of 1 mmolar in PBS (pH 7.4).

[0161] FAB MS: Calc'd for C₄₆H₆₄B₂N₄O₆ [M]⁺ 790; Found [M+1]⁺ 791.

[0162] HPLC: HP 1100 HPLC chromatograph, Waters 5×100 mm NovaPak HR C18column, 0.050 mL injection, 0.75 mL/min, 280 nm detection, A=water (0.1%HFBA) and B=MeCN (0.1% HFBA), gradient 10% B 2 min, 10-80% B over 18min, 80-100% B over 2 min, 100% B 2 min, retention time 13.4 min.

[0163] E. Preparation of Acrylamide Gel ContainingN-[3-(methacrylamido)propyl]-3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonamide(Alizarin Red S Monomer) andα,α′-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-1,4-xylene:

[0164] Ethylene glycol solution containing 30% wt. acrylamide and 0.8%wt. N,N′-methylenebisacrylamide was prepared.N-[3-(methacrylamido)propyl]-3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonamide(1.5 mg, 3.38×10⁻⁶ mole) andα,α′-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-1,4-xylene(28 mg, 3.54×10⁻⁵ mole) were combined with 800 μL of ethylene glycolmonomer solution and 40 μL of 5% wt. aqueous ammonium persulfate. Thisformulation was placed in a glove box purged with nitrogen along with amold constructed from glass microscope slides and 100 micron stainlesssteel spacer. An aqueous solution ofN,N,N′,N′-tetramethylethylenediamine (40 μL, 5% wt.) was added to themonomer solution to accelerate polymerization and the final formulationwas poured into a glass mold. The mold was left under nitrogenatmosphere for 16 hours, after which it was immersed in PBS (pH=7.4) andthe glass slides were separated to afford a hydrogel polymer in a formof a thin film. The resulting hydrogel thin film was washed with 100 mLof phosphate buffered saline containing 1 mM lauryl sulfate sodium saltfor 3 days, the solution being changed every day, followed by washingwith MeOH/PBS (20/80 by vol., 3×100 mL), and finally with PBS (pH=7.4,3×100 mL). Hydrogel polymer was stored in PBS (10 mM PBS, pH=7.4)containing 0.2% wt. sodium azide and 1 mM EDTA sodium salt.

[0165] F. Modulation of Absorbance With Glucose and Lactate

[0166] The modulation of the absorbance of the indicator hydrogel (whichcontains two recognition elements) prepared in this example by glucoseand lactate was determined. The acrylamide gel was mounted in PMMA cellin the same way as described in Example 5. Phosphate buffered saline(PBS), pH=7.4 containing the desired amount of glucose or sodium lactatewas heated to 37° C. in a water bath and placed in the PMMA cellcontaining the gel after which the PMMA cell was allowed to equilibratefor 15 min at 37° C. Absorbance measurement for each glucose or lactateconcentration was conducted in triplicate. For each measurement,absorbance at 650 nm was used as a blank, A(650 nm) was subtracted fromall values of A(450 nm) and A(530 nm).

[0167]FIG. 7 shows the absorbance spectra for acrylamide gel (30%)containing 4 mM Alizarin Red S monomer (1:1000 molar ratio of AlizarinRed:acrylamide) and 44 mM bis boronic acid monomer (1:95 molar ratio ofboronic acid monomer:acrylamide) with and without glucose. FIG. 8 showsthe effect of glucose on absorbance of acrylamide gel (30%) containing 4mM Alizarin Red S monomer and 44 mM bis boronic acid monomer. FIG. 9shows the effect of sodium lactate on absorbance of acrylamide gel (30%)containing 4 mM Alizarin Red S monomer and 44 mM bis boronic acidmonomer. The absorbance of the indicator was affected by the presence ofglucose, but not substantially affected by the presence of lactate.

What is claimed is:
 1. An indicator macromolecule for detecting thepresence or concentration of an analyte in an aqueous environment, saidmacromolecule comprising a copolymer of: a) one or more indicatorcomponent monomers which individually are not sufficiently water solubleto permit their use in an aqueous environment for detecting the presenceor concentration of said analyte; and b) one or more hydrophilicmonomers; such that the macromolecule is capable of detecting thepresence or concentration of said analyte in an aqueous environment. 2.The indicator macromolecule of claim 1, wherein the macromolecule iscapable of detection by an optical change.
 3. The indicatormacromolecule of claim 1, wherein the indicator component monomercomprises an N-(o-boronobenzyl)aminomethylanthracene derivative.
 4. Theindicator macromolecule of claim 3, wherein the indicator componentmonomer is selected from the group consisting of9-[[N-methacryloylaminopropyl-N-(o-boronobenzyl)amino]-methyl]anthracene;9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)-ethylamino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)-propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[3-(methacrylamido)-propylamino]methyl]anthracene;9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)-ethylamino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[2-(2-methacroyloxyethoxy)-ethylamino]methyl]-10-[N-[2-boronobenzyl)]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]anthracene;N-[3-(methacrylamido)propyl]-3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonamide;α,α′-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-1,4-xylene;and salts or derivatives thereof.
 5. The indicator macromolecule ofclaim 3, wherein the hydrophilic monomer comprises[3-(methacryloylamino)-propyl]trimethylammonium chloride.
 6. Theindicator macromolecule of claim 1, wherein the indicator componentmonomer is selected from the group consisting of a lanthanide chelateand a polyaromatic hydrocarbon.
 7. The indicator macromolecule of claim1, wherein the molar ratio of hydrophilic monomer:indicator componentmomomer is from about 2:1 to about 1000:1.
 8. The indicatormacromolecule of claim 7, wherein the ratio of hydrophilicmonomer:indicator component momomer is from about 5:1 to about 50:1. 9.The indicator macromolecule of claim 8, wherein the ratio of hydrophilicmonomer:indicator component momomer is about 5:1.
 10. The indicatormacromolecule of claim 1, wherein the analyte detected is selected fromthe group consisting of a vicinal diol; an α-hydroxy acid, a β-keto acidoxygen; carbon dioxide; zinc, potassium, hydrogen, or carbonate ions; atoxin; a mineral; and a hormone.
 11. The indicator macromolecule ofclaim 10, wherein the analyte detected is a vicinal diol which comprisesa saccharide.
 12. The indicator macromolecule of claim 11, wherein thesaccharide is glucose.
 13. The indicator macromolecule of claim 1,wherein i) the molar ratio of hydrophilic monomer:indicator componentmomomer is from about 2:1 to about 15:1, ii) the indicator componentmonomer comprises an N-(o-boronobenzyl)amino]methyl]anthracenederivative, iii) the hydrophilic monomer comprises[3-(methacryloylamino)propyl]trimethylammonium chloride, and iv) themacromolecule exhibits an excimer effect.
 14. A method for theproduction of an indicator macromolecule for detecting the presence orconcentration of an analyte in an aqueous environment, said methodcomprising copolymerizing: a) one or more indicator component monomerswhich individually are not sufficiently water soluble to permit theiruse in an aqueous environment for detecting the presence orconcentration of said analyte; and b) one or more hydrophilic monomers;such that the resulting macromolecule is capable of detecting thepresence or concentration of said analyte in an aqueous environment. 15.The method of claim 14, wherein the macromolecule is capable ofdetection by an optical change.
 16. The method of claim 14, wherein theindicator component monomer comprises anN-(o-boronobenzyl)aminomethylanthracene derivative.
 17. The method ofclaim 16, wherein the indicator component monomer is selected from thegroup consisting of9-[[N-methacryloylaminopropyl-N-(o-boronobenzyl)amino]-methyl]anthracene;9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)-ethylamino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)-propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[3-(methacrylamido)-propylamino]methyl]anthracene;9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)-ethylamino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[2-(2-methacroyloxyethoxy)-ethylamino]methyl]-10-[N-[2-boronobenzyl)]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]anthracene;N-[3-(methacrylamido)propyl]-3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonamide;α,α′-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-1,4-xylene;and salts or derivatives thereof.
 18. The method of claim 14, whereinthe hydrophilic monomer comprises[3-(methacryloylamino)-propyl]trimethylammonium chloride.
 19. The methodof claim 14, wherein the indicator component monomer is selected fromthe group consisting of a lanthanide chelate and a polyaromatichydrocarbon.
 20. The method of claim 14, wherein the molar ratio ofhydrophilic monomer:indicator component momomer is from about 2:1 toabout 1000:1.
 21. The method of claim 20, wherein the ratio ofhydrophilic monomer:indicator component momomer is from about 5:1 toabout 50:1.
 22. The method of claim 21, wherein the ratio of hydrophilicmonomer:indicator component momomer is about 5:1.
 23. The method ofclaim 14, wherein the analyte detected is selected from the groupconsisting of a vicinal diol; an α-hydroxy acid; a β-keto acid; oxygen;carbon dioxide; zinc, potassium, hydrogen, or carbonate ions; a toxin; amineral; and a hormone.
 24. The method of claim 23, wherein the analytedetected is a vicinal diol which comprises a saccharide.
 25. The methodof claim 24, wherein the saccharide is glucose.
 26. The method of claim14, wherein i) the molar ratio of hydrophilic monomer:indicatorcomponent momomer is from about 2:1 to about 15:1, ii) the indicatorcomponent monomer comprises an N-(o-boronobenzyl)aminomethylanthracenederivative, iii) the hydrophilic monomer comprises[3-(methacryloylamino)propyl]trimethylammonium chloride, and iv) themacromolecule exhibits an excimer effect.
 27. A method for detecting thepresence or concentration of an analyte in a sample having an aqueousenvironment, said method comprising: a) exposing the sample to anindicator macromolecule, said macromolecule comprising a copolymer of:i) one or more indicator component monomers which individually are notsufficiently water soluble to permit their use in an aqueous environmentfor detecting the presence or concentration of said analyte; and ii) oneor more hydrophilic monomers; such that the resulting macromolecule iscapable of detecting the presence or concentration of said analyte in anaqueous environment, and wherein the indicator macromolecule has adetectable quality that changes in a concentration-dependent manner whensaid macromolecule is exposed to said analyte; and b) measuring anychange in said detectable quality to thereby determine the presence orconcentration of said analyte in said sample.
 28. The method of claim27, wherein the change in said detectable quality is an optical change.29. The method of claim 27, wherein the indicator component monomercomprises an N-(o-boronobenzyl)aminomethylanthracene derivative.
 30. Themethod of claim 29, wherein the indicator component monomer is selectedfrom the group consisting of9-[[N-methacryloylaminopropyl-N-(o-boronobenzyl)amino]-methyl]anthracene;9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)-ethylamino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)-propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[3-(methacrylamido)-propylamino]methylanthracene;9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)-ethylamino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[2-(2-methacroyloxyethoxy)-ethylamino]methyl]-10-[N-[2--boronobenzyl)]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]anthracene;N-[3-(methacrylamido)propyl]-3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonamide;α,α′-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-1,4-xylene;and salts or derivatives thereof.
 31. The method of claim 27, whereinthe hydrophilic monomer comprises[3-(methacryloylamino)-propyl]trimethylammonium chloride.
 32. The methodof claim 27, wherein the indicator component monomer is selected fromthe group consisting of a lanthanide chelate and a polyaromatichydrocarbon.
 33. The method of claim 27 wherein the molar ratio ofhydrophilic monomer:indicator component momomer is from about 2:1 toabout 1000:1.
 34. The method of claim 33, wherein the ratio ofhydrophilic monomer:indicator component momomer is from about 5:1 toabout 50:1.
 35. The method of claim 34 wherein the ratio of hydrophilicmonomer:indicator component momomer is about 5:1.
 36. The method ofclaim 27, wherein the analyte detected is selected from the groupconsisting of a saccharide; oxygen; carbon dioxide; and zinc, potassium,hydrogen, or carbonate ions.
 37. The method of claim 36, wherein theanalyte detected is a saccharide.
 38. The method of claim 37, whereinthe saccharide is glucose.
 39. The method of claim 27, wherein i) themolar ratio of hydrophilic monomer:indicator component momomer is fromabout 2:1 to about 15:1, ii) the indicator component monomer comprisesan N-(o-boronobenzyl)aminomethylanthracene derivative, iii) thehydrophilic monomer comprises[3-(methacryloylamino)propyl]trimethylammonium chloride, and iv) themacromolecule exhibits an excimer effect.
 40. The method of claim 39,wherein said macromolecule serves as both an indicator and a reference.41. A macromolecule which is capable of exhibiting an excimer effect,which comprises a copolymer of: a) one or more excimer forming monomers,the molecules of which are capable of exhibiting an excimer effect whensuitably oriented with respect to each other; and b) one or more othermonomers; such that the resulting macromolecule exhibits said excimereffect.
 42. The macromolecule of claim 41, wherein the macromolecule iscapable of detecting the presence or concentration of an analyte. 43.The macromolecule of claim 42, wherein a) the excimer forming monomerindividually is not sufficiently water soluble to permit its use in anaqueous environment for detecting the presence or concentration of saidanalyte; and b) the other monomer is a hydrophilic monomer; such thatthe macromolecule is capable of detecting the presence or concentrationof said analyte in an aqueous environment.
 44. The macromolecule ofclaim 42, wherein the excimer effect does not substantially change inresponse to changes in the presence or concentration of the analyte. 45.The macromolecule of claim 44, wherein i) the molar ratio of othermonomer:excimer forming momomer is from about 2:1 to about 15:1, ii) theexcimer forming monomer comprises anN-(o-boronobenzyl)aminomethylanthracene derivative, and iii) the othermonomer comprises [3-(methacryloylamino)propyl]trimethylammoniumchloride.
 46. The macromolecule of claim 41, wherein the excimer formingmonomer is selected from the group consisting of a lanthanide chelateand a polyaromatic hydrocarbon.
 47. The macromolecule of claim 45,wherein the excimer forming monomer is selected from the groupconsisting of9-[[N-methacryloylaminopropyl-N-(o-boronobenzyl)amino]-methyl]anthracene;9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)-ethylamino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)-propylamino]methyl]-10-[N-(2-boronbenzyl)-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[3-(methacrylamido)-propylamino]methylanthracene;9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)-ethylamino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[2-(2-methacroyloxyethoxy)-ethylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(2-hydroxyethoxy)9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]anthracene;N-[3-(methacrylamido)propyl]-3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonamide;α,α′-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-1,4-xylene;and salts or derivatives thereof.
 48. A method for producing amacromolecule which is capable of exhibiting an excimer effect, whichmethod comprises copolymerizing: a) one or more excimer formingmonomers, the molecules of which are capable of exhibiting an excimereffect when suitably oriented with respect to each other; and b) one ormore other monomers; such that the resulting macromolecule exhibits saidexcimer effect.
 49. The method of claim 48, wherein the macromolecule iscapable of detecting the presence or concentration of an analyte. 50.The method of claim 49, wherein a) the excimer forming monomerindividually is not sufficiently water soluble to permit its use in anaqueous environment for detecting the presence or concentration of saidanalyte; and b) the other monomer is a hydrophilic monomer; such thatthe macromolecule is capable of detecting the presence or concentrationof said analyte in an aqueous environment.
 51. The method of claim 49,wherein the excimer effect does not substantially change in response tochanges in the presence or concentration of the analyte.
 52. The methodof claim 51, wherein i) the molar ratio of other monomer:excimer formingmomomer is from about 2:1 to about 15:1, ii) the excimer forming monomercomprises an N-(o-boronobenzyl)aminomethylanthracene derivative, andiii) the other monomer comprises[3-(methacryloylamino)propyl]trimethylammonium chloride.
 53. The methodof claim 48, wherein the excimer forming monomer is selected from thegroup consisting of a lanthanide chelate and a polyaromatic hydrocarbon.54. The method of claim 52, wherein the excimer forming monomer isselected from the group consisting of9-[[N-methacryloylaminopropyl-N-(o-boronobenzyl)amino]-methyl]anthracene;9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)-ethylamino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)-propylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[3-(methacrylamido)-propylamino]methyl]anthracene;9-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)-ethylamino]methyl]anthracene;9-[N-(2-boronobenzyl)-N-[2-(2-methacroyloxyethoxy)-ethylamino]methyl]-10-[N-(2-boronobenzyl)-N-[2-(2-hydroxyethoxy)ethylamino]methyl]-anthracene;9,10-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]anthracene;9,10-bis[N-(2-boronobenzyl)-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]anthracene;N-[3-(methacrylamido)propyl]-3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonamide;α,α′-bis[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-1,4-xylene;and salts or derivatives thereof.
 55. A method for detecting thepresence or concentration of an analyte in a sample, said methodcomprising: a) exposing the sample to an indicator macromolecule, saidmacromolecule comprising a copolymer of: i) one or more indicatorcomponent monomers, the molecules of which are capable of exhibiting anexcimer effect when suitably oriented with respect to each other, andwhich are also capable of detecting the presence or concentration of ananalyte; and ii) one or more other monomers; such that the resultingmacromolecule exhibits said excimer effect, and wherein the indicatormacromolecule has a detectable quality that changes in aconcentration-dependent manner when said macromolecule is exposed tosaid analyte; and b) measuring any change in said detectable quality tothereby determine the presence or concentration of said analyte in saidsample.
 56. The method of claim 55, wherein the excimer effect does notsubstantially change in response to changes in the presence orconcentration of the analyte.
 57. The method of claim 56, wherein i) themolar ratio of other monomer:indicator component momomer is from about2:1 to about 15:1, ii) the indicator component monomer comprises anN-(o-boronobenzyl)amino]methyl]anthracene derivative, and iii) the othermonomer comprises [3-(methacryloylamino)propyl]trimethylammoniumchloride.
 58. The method of claim 55, wherein the indicator componentmonomer is selected from the group consisting of a lanthanide chelateand a polyaromatic hydrocarbon.
 59. The method of claim 57, wherein theindicator component monomer is selected from the group consisting of9-[[N-methacryloylaminopropyl-N-(o-boronobenzyl)amino]-methyl]anthracene;9-[N-(2-boronobenzyl)-N-[3-(methacrylamido)-propylamino]methyl]-10-[N-[2-(5,5-dimethylborinan-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]-methyl]anthracene;9-[N-(2-boronobenzyl)-N-[2-(2-methacroyloxyethoxy)-ethylamino]-methyl]-10-[N-(2-boronobenzyl)-N-[2-(2-hydroxyethoxy)ethylamino]methyl]-anthracene;and9,10-bis[N-(2-boronobenzyl)-N-[2-(2-methacroyloxyethoxy)ethylamino]methyl]anthracene;and salts or derivatives thereof.